The volume-regulated anion channel as a regulator of secretory activity in the intestinal epithelium

项目总结 肠上皮依靠广泛的细胞-细胞信号来维持吸收和吸收之间的紧密平衡。 和分泌活动，但这一信号的错综复杂仍未确定。我们最近发现， 轮状病毒是全球威胁生命的腹泻的最常见原因，感染轮状病毒的细胞释放数百 二磷酸腺苷(ADP)的脉冲引起细胞间钙波，使未感染的细胞失去调节。 阻断ADP受体P2Y1显著降低轮状病毒感染小鼠的腹泻严重程度，提示 这种信号在调节分泌活动中是不可或缺的。而轮状病毒触发的机制 ADP的释放尚未得到证实，我们的初步数据显示它依赖于容量的激活- 调节阴离子通道(Vrac)。Vrac越来越被认为是旁分泌信号的管道。 与多种细胞内应激源有关，但尚未在上皮内进行研究。此外， 在任何情况下调节其激活的机制仍然知之甚少。因此，这样做的目的是 建议使用轮状病毒作为工具来研究vrac在肠道上皮细胞中的作用和激活。vt.给出 轮状病毒非结构蛋白击倒抑制轮状病毒诱导的细胞间钙波 4(Nsp4)，一种钙离子传导通道，我的中心假设是nsp4诱导胞浆内 CA2激活宿主通路，通过vrac激活ADP释放，放大分泌活动 遍及整个上皮细胞。我将结合长期的活体钙成像来验证这一假说。 使用新型荧光Rho生物传感器和基于CRISPR-Cas9的基因编辑来[目标1]确定 RV感染细胞中介导vrac激活的细胞内途径。给定的ADP释放减少 Rho激酶的药理抑制或钙增强型轮状病毒蛋白nsp4的敲除 推测钙和Rho都参与了vrac的激活。使用人的肠类和 小鼠轮状病毒腹泻模型，我将[目的2]确定vrac激活对小鼠腹水分泌活性的影响。 肠上皮细胞。这项工作将确定治疗分泌性腹泻的新靶点， 这是全球儿童死亡的主要原因。此外，我们的发现将解决知识差距问题 周围vrac的激活及其在旁分泌信号中的作用。鉴于vrac卷入了一场 越来越多的病理过程，使用这种临床相关的方法将产生信息 可翻译为人类健康和其他科学学科。

PROJECT SUMMARY The intestinal epithelium relies on extensive cell-cell signaling to maintain a tight balance between absorptive and secretory activity, but the intricacies of this signaling remain uncharacterized. We recently discovered that cells infected with rotavirus, the most common cause of life-threatening diarrhea worldwide, release hundreds of pulses of adenosine diphosphate (ADP) causing intercellular calcium waves that dysregulate uninfected cells. Blocking the ADP receptor P2Y1 significantly reduces diarrhea severity in rotavirus-infected mice, suggesting that this signaling is integral in regulating secretory activity. While the mechanism through which rotavirus triggers the release of ADP has not been confirmed, our preliminary data show it relies on activation of the volume- regulated anion channel (VRAC). VRAC is increasingly recognized as a conduit for paracrine signals associated with a variety of intracellular stressors, but it has yet to be studied within the epithelium. Further, the mechanisms regulating its activation in any context remain poorly understood. Therefore, the objective of this proposal is to use rotavirus as a tool to study the role and activation of VRAC in the intestinal epithelium. Given that rotavirus-induced intercellular calcium waves are inhibited by knock down of rotavirus non-structural protein 4 (NSP4), a Ca2+-conducting channel, my central hypothesis is that NSP4-induced elevations of cytosolic Ca2+ activate host pathways that trigger ADP release through VRAC, amplifying secretory activity throughout the epithelium. I will test this hypothesis by using long-term live calcium imaging in conjunction with novel fluorescent Rho biosensors and CRISPR-Cas9 based gene editing to [Aim 1] determine the intracellular pathway mediating VRAC activation in RV-infected cells. Given ADP release is reduced upon pharmacological inhibition of Rho kinase or knockdown of the calcium-augmenting rotavirus protein NSP4, we expect both calcium and Rho are involved in VRAC activation. Using both human intestinal enteroids and the mouse model of rotavirus diarrhea, I will [Aim 2] determine the effect of VRAC activation on secretory activity in the intestinal epithelium. This work will identify novel therapeutic targets for the treatment of secretory diarrhea, a leading cause of mortality among children worldwide. Furthermore, our findings will address gaps in knowledge surrounding VRAC activation and its role in paracrine signaling. Given that VRAC has been implicated in an increasing variety of pathological processes, using this clinically relevant approach will yield information translatable to both human health and other scientific disciplines.